Project Summary/Abstract: RNAs play diverse roles in biology, but our ability to fully understand their cellular roles is limited by the lack of tools to study and manipulate RNA. Non-coding RNAs have emerged as key regulators in cellular biology and include both small interfering RNAs and micro-RNAs that regulate mRNA expression through Argonaute- dependent RNA-degradation and thousands of long-intergenic noncoding RNAs (lincRNAs), which generally serve to modulate gene expression, epigenetic states, and post-transcriptional processes. The mammalian genome encodes tens of thousands of lincRNAs and while they have diverse regulatory roles in gene expression, especially in both the positive and negative directions in cancer, it is unknown whether lincRNAs play a significant role in cancer resistance. Although remarkable clinical effects are seen with the BRAF- inhibitor vemurafenib in melanoma, they nevertheless fail due to drug resistance. Genome-wide gain- and loss- of-function pooled screening have revealed many of the genes involved in vemurafenib resistance using in vitro cell line models of melanoma, but the role of lincRNAs in melanoma resistance is still unclear. While many RNA-targeting tools exist to study these processes, none are ideal. For instance, siRNAs suffer from poor specificity and are incapable of targeting nuclear RNAs, such as lincRNAs. Recently, we computationally discovered novel CRISPR-associated enzymes and identified a new family, C2c2, which had significant conservation of two HEPN endoribonuclease domains, suggesting the likely ability for C2c2 to cleave RNAs. I will combine biochemical and bacterial genetic techniques to functionally characterize C2c2, engineer the enzyme for mammalian applications, and use this tool for the interrogation of non-coding RNA mediators of resistance in melanoma. In Aim 1, I will biochemically characterize C2c2 from Leptotrichia shahii and identify the key active residues for RNA cleavage. Validation of reprogrammable RNA targeting will be demonstrated both in vitro using purified protein and in vivo using E. coli. In Aim 2, I will screen 12 C2c2 orthologs to determine the most active RNA-targeting enzyme. I will then express human codon-optimized versions of the top orthologs in mammalian cells to demonstrate endogenous RNA cleavage and perform whole-transcriptome RNA- sequencing to evaluate the specificity of C2c2. In Aim 3, I will develop a genome-wide lincRNA library of C2c2 crRNAs and perform a knockdown screen in a melanoma cell line subjected to BRAF-inhibitor drug selection. I will validate the hits and demonstrate that they are capable of modulating sensitivity to BRAF-inhibitors. This project will develop a reprogrammable RNA-targeting tool and investigate the roles of non-coding RNAs in melanoma resistance. The availability of RNA-targeting tool is useful for many applications and the understanding of non-coding interactions in melanoma resistance is essential for developing better therapies.